EMSA Electrophoretic Mobility Gel Shift Reaction Protocol

EMSA Electrophoretic Mobility Gel Shift Reaction Protocol

EMSA Electrophoretic Mobility Gel Shift Reaction Protocol

Observing a Gel Shift on the Polyacrylamide Gel

• 1. Prepare gel as follows:

1. 10.5 ml of Acrylamide/Bisacrylamide solution
2. 2.5 ml of 10X TGOE buffer
3. 1.75 ml of 50% Glycerol
4. 20.9 ml of ddH2O
5. 0.3 ml of 10% Ammonium Persulfate 30 l of TEMED

• 2. Cast the gel and set it in a cold room (4°C) approximately 5 to 10 min prior to loading the sample
• 3. Wash the wells several times with 1 X TGOE (running buffer).
• 4. Pre-run the gel at 160 V for 15 minutes.
• 5. Wash the wells out several times with 1 X TGOE again.
• 6. Add the optimal volume of Loading Buffer to the samples (the ratio of sample to Loading Buffer should be determined empirically to ensure adequate dilution of the protein/DNA complex).
• 7. Begin the electrophoresis at 160 V.
• 8. Load the samples onto the wells of the gel. Start the gel immediately after loading the samples.
• 9. Electrophorese for approximately 45 min.
• 10. After completion of the electrophoresis, remove the gel and analyze the band shift by Autoradiography

Solutions and Buffers

• Loading buffer 1X TGOE
• 0.05% Bromophenol Blue (see Tip 3)
• 35% (v/v) Glycerol

Gel Buffer 1X TGOE


50% (v/v) Glycerol

Acrylamide/Bisacrylamide Solution CAUTION! See Tip 2
0.33% (w/v) Bisacrylamide
20% (w/v) Acrylamide
Prepare in ddH2O just before use


TGOE Buffer (10X) pH 8.2 using Acetic Acid.
0.25 M Tris
1.9 M Glycine


Binding Buffer (5X) OPTIONAL: add 25 μl of saturated Bromophenol Blue (approximately 0.1% in ddH2O per ml of 5X Binding Buffer (BioRad) (see Tip 3)
25 mM MgCl2
500 μg/ml BSA
300 mM KCl
Store buffer at -70°C.
20% (v/v) Glycerol
100 mM Tris-HCl pH 8.0


poly-[dG-dC]-Deoxyribonucleic Acid

Protein Solution Proteins are typically stable during multiple repeated freeze-thaw cycles.
Thaw proteins quickly on ice.
(X) mg/ml protein (see Tip 1 ) in Protein Dilution Buffer.


Ammonium Persulfate 10% (w/v) Ammonium Persulfate
Prepare in ddH2O just before use


Protein Dilution Buffer 1 mM DTT
150 mM KCl
10% (v/v) Glycerol
50 μg/ml Bovine Serum Albumin
20 mM Tris pH 7.9
Store Dilution Buffer at -70°C.


0.5M DDT Prepare in ddH2O just before use


0.1M DDT Prepare in ddH2O just before use

Materials

• Bromophenol Blue
• TEMED
• Glycerol
• Dry Ice
• Ammonium Persulfate
• Tris
• Potassium Chloride
• Acrylamide
• Magnesium Chloride
• Acetic Acid
• p[dG-dC]
• DTT
• Glycine
• Poly [dG-dC]
• Bisacrylamide
• Bovine Serum Albumin

Protocol Tips:

1. The amount of protein added must be determined empirically and will be based on the affinity of the protein to bind to the DNA

2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

3. Bromophenol Blue may interfere with protein binding to DNA. The effects of the addition of Bromophenol Blue should be determined empirically.